Biotechnology
| Poster #543 | |
| » | Abstract |
| » | Introduction |
| » | Materials and Methods |
| » | Two-Color Western Blots |
| » | EMSA Using IR Dye Labeling |
| » | Conclusions |
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Use of Infrared Fluorescent Technology for Analysis of Signal Transduction and Protein-Nucleic Acid Interactions
Huaxian Chen*, Marjeta Urh**, Amy Schutz and D.
Michael Olive
LI-COR Inc., 4647 Superior Street, Lincoln, NE 68504
* hchen@licor.com, **murh@licor.com
Abstract
Cellular signaling processes are regulated by a number of molecular interactions. These include the binding of proteins to key regulatory sites on DNA as well as phosphorylation of protein receptors, kinases, and transcription factors. The analysis of these processes currently relies on the use of radioisotopic or chemiluminescent-based methods, neither of which gives optimal results. We have developed methods employing two-color infrared fluorescent technology for the analysis of signal transduction events, as well as electrophoretic mobility shift assays (EMSA) for monitoring protein-DNA binding. Lysates of human cultured cells that were either unstimulated or specifically stimulated were examined for either phosphorylated IFNY receptor or activated MAP kinase using two different primary antibodies, one to an epitope common to both the active and inactive forms of the protein and one specific for the activated protein.
Detection was performed using fluorescent-labeled secondary antibodies coupled to either IRDye® 700 infrared dye, IRDye® 800 infrared dye, Cy5.5 or Alexa680 dyes. In both cases, phosphorylated and total amounts of the IFNY receptor or MAP kinase could be visualized and quantitated simultaneously without the need for stripping and reprobing. A model system for assessing protein-nucleic acid interactions by EMSA was developed based on the binding of T7 RNA polymerase to its promoter. Varying amounts of protein were combined with a PCR-generated IRDye® 800 infrared dye-labeled DNA fragment. Protein binding could be monitored quantitatively following scanning of the gel on the Odyssey™ Infrared Imager. Near infrared technology enables sensitive, precise monitoring of signal transduction and protein-nucleic acid regulatory binding events due to the low background fluorescence of membranes and gels in this spectral region. Furthermore, two-color capability facilitates the simultaneous analysis of phosphorylation events without the additional labor and decreased precision associated with stripping and reprobing membranes analyzed in a single color.
